Project Summary Trypanosoma brucei is a single cell protozoan parasite that causes human African Sleeping Sickness, that is fatal if untreated. Related trypanosomatid pathogens, including numerous Leishmania species, cause even more morbidity and mortality worldwide. The regulation of gene expression is essential for parasite development and survival in their sequential host environments. Exactly how trypanosomatid gene expression is regulated is currently not well understood, but the relative contribution of transcriptional control was thought to be absent. We have demonstrated a role for a modified DNA base, called base J, and histone H3 variant (H3.V) in the regulation of RNA polymerase II (Pol II) transcription termination in T. brucei and L. major. This includes sites within polycistronic gene clusters leading to ?premature? termination and silencing of genes downstream. Our long-term goal is to dissect epigenetic mechanisms of gene expression in trypanosomatids. The objective of this grant is to determine the mechanism of J/H3.V regulated Pol II transcription termination in T. brucei. The central hypothesis is that base J/H3V regulates termination via regulating specific stages of a ?torpedo-PP1? termination mechanism. The torpedo component involves 5'-3' exonuclease (XRND), helicase (Sen1) and an RNA binding protein (RBP33). Pol II pausing and stimulation of the torpedo mechanism in other eukayotes involves dephosphorylation of the C-terminal domain of Pol II by PP1 protein phosphatase as part of the multimeric PTW/PP1 complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and PP1. We have identified a similar complex in kinetoplastids, called PJJW/PP1, that replaced Tox4 with a J-binding protein (JBP3). These data led to the current model where base J (via JBP3) recruits the PJJW/PP1 complex at termination sites and PP1 dephosphorylates Pol II CTD, pausing elongation allowing the torpedo mechanism to efficiently dissociate Pol II. Our specific aims will test the following hypothesis: Aim 1 will directly test our torpedo model by assessing the role of these protein factors in transcription termination in T. brucei. We will then analyze the function of individual protein components using a combination of in vitro and in vivo analyses. Aim 2 will characterize the PJJW/PP1 complex, including the roles of PNUTS and JBP3 as the assembly and chromatin recruitment factor, respectively. Aim 3 will elucidate the role of the PJJW/PP1 complex in termination, including the function of PP1 in modifying the CTD of pol II. The proposed work will provide a fundamental understanding trypanosomatid gene expression, yielding a comprehensive view of the role of transcriptional control and clues to it mechanisms. These studies may prove useful in identifying novel approaches to prevention, treatment and diagnosis of the debilitating and deadly diseases caused by these parasites. ! ! ! !